Tohoku
J. Exp.
Erect Radical
Med., 1992,
167, 185-188
of Omeprazole
on Ascorbate
Free
Formation
TADASHIOHARA,RIKUKOSASAKI*,DAISUKESHIBUYAt, SHIGERU ASAKI and TAKAYOSHI TOYOTA Department of Internal Gastroenterology,Sendai Shakai Hoken Hospital, Sendai 981, *Department of Internal Medicine, Miyagi Kenko-Hoken Hospital, Sendai 982, 'Department of Internal Gastroenterology , Sendai City Hospital, Sendai 980, and $the Third Department of Internal Medicine, Tohoku University School of Medicine, Sendai 980 OHARA,T., SASAKI,R., SHIBUYA,D., ASAKI,S. and TOYOTA,T. Effect of Omeprazole on Ascorbate Free Radical Formation. Tohoku J. Exp. Med., 1992, 167 (3), 185-188 In order to study the effect of omeprazole on the K/ [H+] value of ascorbic acid (K the ascorbic acid-ascorbic free radical equilibrium constant), changes of the concentration of ascorbate free radical in guinea pig sera were examined after intraperitoneal administration of omeprazole. Furthermore, to see the in vitro effect of omeprazole on ascorbate free radical, changes were examined in ascorbate solutions after addition of omeprazole. It was found that the K/ [H+] value in the serum increased significantly after administration of omeprazole and also that the drug amplified the electron spin resonance (ESR) signal intensity of ascorbate free radical in vitro. These results suggested that omeprazole acts like oxygen radicals. omeprazole ; ascorbic acid ; ascorbate radical ; oxygen radicals
Omeprazole is currently used to treat intractable peptic ulcer resistant to 112 blokers. Omeprazole has been reported to be a proton pump inhibitor, which potently blocks gastric secretion (Olbe et al. 1983). Recent experimental studies have shown, however, that omeprazole administration causes a histological change as vacuolar degeneration of parietal cells in rat gastric mucosa (Karasawa et al. 1989; Helander et al. 1990). Karasawa et al. (1989) deduced that lipid peroxidation is linked to the vacuolar degeneration of parietal cells. It is necessary to study the effect of omeprazole from the standpoint of free radicals, because the lipid peroxidation is triggered by radicals such as suproxide anion (OZ) in intracellular and extracellular fluids. We have previously reported that there is an apparent equilibrium between the reduced (RAsA) and oxidized (DAsA) forms of ascorbic acid and ascorbate free radicals (AFR) in human serum. Since RAsA + DAsA = 2AFR + H+, K/ ReceivedMay 14, 1992; revision acceptedfor publication June 16, 1992. 185
186
T. Ohara
et al.
[H+] can be calculated from [AFR]2/( [RAsA][DAsA]) (K=the equilibrium constant). The K/ [H+] value is constant in healthy subjects regardless of total ascorbic acid concentration, age, and sex (Sasaki et al. 1983). The K/ [H+] values have been reported to be 4.5 X 10-9-5.4 X 10-9 for ascorbic acid solution (Foester et al. 1965) and 0.18 X 10-6-4.85 X 10-6 (the mean, 1.08 X 10-6) for normal human and guinea pig serum (Sasaki et al. 1984) ; the K/ [H+] value in serum is exceedingly larger than that in ascorbic acid solution. In serum are many factors affecting AFR content and possibly biassing the equibrium towards the larger K/ [H+] value (Sasaki et al. 1985). While the K/ [H+] value is constant in healthy controls, it has been found to be increased in many desease states (Sasaki et al. 1984). Since the increase of K/ [H+] is resulted from the increase of AFR content, RAsA should function as a scavenger of oxygen radicals such as 02 , hydroxy radical (.011) and singlet oxygen ('02). In fact, we have observed that the K/ [H+] value is increased by addition of 02 into serum in vitro (Sasaki et al. 1989). The present study reports the effects of omeprazole on the K/ [H+] value of guinea pig serum as well as on the AFR concentration in ascorbate solution, and discuss a possibility that omeprazole has an influence upon the oxidation state of ascorbate in the serum. MATERIALS AND METHODS Ten male Hartley guinea pigs, weighing between 400 and 450 g, were used. Animals were allowed for free access to a standard diet and water. Their access to food, but not water, was stopped 15 hr before experiment. An omeprazole solution (6 mg/ml) was made by dissolving in 40% polyethylene glycol containing 0.5% sodium bicarbonate (solvent A). The solution was administered intraperitoneally to guinea pigs (20 mg/kg) and blood was drawn from femoral vein after 30 min. Control animals were given 1 ml of physiological saline intraperitoneally. K/ [H+] values were calculated as previously reported (Sasaki et al. 1984). To see the in vitro effect of omeprazole, changes in ESR signal intensity of ARF were examined with the following solutions as previously described (Sasaki et al. 1985) : (1) 10 M ascorbate solution (control) ; (2) 0.1 ml of 10_3M ascorbate solution added with 0.1 ml of solvent A and 0.8 ml of phosphate buffer, pH 7.4 (ascorbate solution plus solvent A) ; (3) 0.1 ml of 10 M ascorbate solution added with 0.1 ml of the omeprazole solution and 0.8 ml of phosphate buffer, pH
TABLE1. Comparison of the K/[H] omeprazole-treated animals
values in sera of the control and
Effect
Fig. 1.
Changes
of Omeprazole
in ESR
signal
on the K Value
intensity
of Ascorbic
Acid
187
of AFR.
ESR spectrum of free radical observed in human serum at room temperature. The signals at the both ends are due to manganese ion. The g-value was 2.0054 and the hyperfine
splitting
constant,
1.84 gauss.
OPZ denotes
omepr-
azole.
7.4 (ascorbate solution plus the omeprazole solution). ESR measurements were made with a Varian E-109 EPR spectrometer operated at X-band, and 105Hz modulation at room temperature. To obtain a good signal-to-noise ratio,an aqueous flat cell made of quartz was used. The ESR spectra were recorded under the conditions of 10 mW microwave power and one gauss modulation amplitude. The g-value of the free radical was determined using peroxylamine disulfonate ion radical (g=2.0054) as a standard. Results were presented as the mean ±s.D. Student's t-test was used for statistical analysis and p
J. Exp.
Erect Radical
Med., 1992,
167, 185-188
of Omeprazole
on Ascorbate
Free
Formation
TADASHIOHARA,RIKUKOSASAKI*,DAISUKESHIBUYAt, SHIGERU ASAKI and TAKAYOSHI TOYOTA Department of Internal Gastroenterology,Sendai Shakai Hoken Hospital, Sendai 981, *Department of Internal Medicine, Miyagi Kenko-Hoken Hospital, Sendai 982, 'Department of Internal Gastroenterology , Sendai City Hospital, Sendai 980, and $the Third Department of Internal Medicine, Tohoku University School of Medicine, Sendai 980 OHARA,T., SASAKI,R., SHIBUYA,D., ASAKI,S. and TOYOTA,T. Effect of Omeprazole on Ascorbate Free Radical Formation. Tohoku J. Exp. Med., 1992, 167 (3), 185-188 In order to study the effect of omeprazole on the K/ [H+] value of ascorbic acid (K the ascorbic acid-ascorbic free radical equilibrium constant), changes of the concentration of ascorbate free radical in guinea pig sera were examined after intraperitoneal administration of omeprazole. Furthermore, to see the in vitro effect of omeprazole on ascorbate free radical, changes were examined in ascorbate solutions after addition of omeprazole. It was found that the K/ [H+] value in the serum increased significantly after administration of omeprazole and also that the drug amplified the electron spin resonance (ESR) signal intensity of ascorbate free radical in vitro. These results suggested that omeprazole acts like oxygen radicals. omeprazole ; ascorbic acid ; ascorbate radical ; oxygen radicals
Omeprazole is currently used to treat intractable peptic ulcer resistant to 112 blokers. Omeprazole has been reported to be a proton pump inhibitor, which potently blocks gastric secretion (Olbe et al. 1983). Recent experimental studies have shown, however, that omeprazole administration causes a histological change as vacuolar degeneration of parietal cells in rat gastric mucosa (Karasawa et al. 1989; Helander et al. 1990). Karasawa et al. (1989) deduced that lipid peroxidation is linked to the vacuolar degeneration of parietal cells. It is necessary to study the effect of omeprazole from the standpoint of free radicals, because the lipid peroxidation is triggered by radicals such as suproxide anion (OZ) in intracellular and extracellular fluids. We have previously reported that there is an apparent equilibrium between the reduced (RAsA) and oxidized (DAsA) forms of ascorbic acid and ascorbate free radicals (AFR) in human serum. Since RAsA + DAsA = 2AFR + H+, K/ ReceivedMay 14, 1992; revision acceptedfor publication June 16, 1992. 185
186
T. Ohara
et al.
[H+] can be calculated from [AFR]2/( [RAsA][DAsA]) (K=the equilibrium constant). The K/ [H+] value is constant in healthy subjects regardless of total ascorbic acid concentration, age, and sex (Sasaki et al. 1983). The K/ [H+] values have been reported to be 4.5 X 10-9-5.4 X 10-9 for ascorbic acid solution (Foester et al. 1965) and 0.18 X 10-6-4.85 X 10-6 (the mean, 1.08 X 10-6) for normal human and guinea pig serum (Sasaki et al. 1984) ; the K/ [H+] value in serum is exceedingly larger than that in ascorbic acid solution. In serum are many factors affecting AFR content and possibly biassing the equibrium towards the larger K/ [H+] value (Sasaki et al. 1985). While the K/ [H+] value is constant in healthy controls, it has been found to be increased in many desease states (Sasaki et al. 1984). Since the increase of K/ [H+] is resulted from the increase of AFR content, RAsA should function as a scavenger of oxygen radicals such as 02 , hydroxy radical (.011) and singlet oxygen ('02). In fact, we have observed that the K/ [H+] value is increased by addition of 02 into serum in vitro (Sasaki et al. 1989). The present study reports the effects of omeprazole on the K/ [H+] value of guinea pig serum as well as on the AFR concentration in ascorbate solution, and discuss a possibility that omeprazole has an influence upon the oxidation state of ascorbate in the serum. MATERIALS AND METHODS Ten male Hartley guinea pigs, weighing between 400 and 450 g, were used. Animals were allowed for free access to a standard diet and water. Their access to food, but not water, was stopped 15 hr before experiment. An omeprazole solution (6 mg/ml) was made by dissolving in 40% polyethylene glycol containing 0.5% sodium bicarbonate (solvent A). The solution was administered intraperitoneally to guinea pigs (20 mg/kg) and blood was drawn from femoral vein after 30 min. Control animals were given 1 ml of physiological saline intraperitoneally. K/ [H+] values were calculated as previously reported (Sasaki et al. 1984). To see the in vitro effect of omeprazole, changes in ESR signal intensity of ARF were examined with the following solutions as previously described (Sasaki et al. 1985) : (1) 10 M ascorbate solution (control) ; (2) 0.1 ml of 10_3M ascorbate solution added with 0.1 ml of solvent A and 0.8 ml of phosphate buffer, pH 7.4 (ascorbate solution plus solvent A) ; (3) 0.1 ml of 10 M ascorbate solution added with 0.1 ml of the omeprazole solution and 0.8 ml of phosphate buffer, pH
TABLE1. Comparison of the K/[H] omeprazole-treated animals
values in sera of the control and
Effect
Fig. 1.
Changes
of Omeprazole
in ESR
signal
on the K Value
intensity
of Ascorbic
Acid
187
of AFR.
ESR spectrum of free radical observed in human serum at room temperature. The signals at the both ends are due to manganese ion. The g-value was 2.0054 and the hyperfine
splitting
constant,
1.84 gauss.
OPZ denotes
omepr-
azole.
7.4 (ascorbate solution plus the omeprazole solution). ESR measurements were made with a Varian E-109 EPR spectrometer operated at X-band, and 105Hz modulation at room temperature. To obtain a good signal-to-noise ratio,an aqueous flat cell made of quartz was used. The ESR spectra were recorded under the conditions of 10 mW microwave power and one gauss modulation amplitude. The g-value of the free radical was determined using peroxylamine disulfonate ion radical (g=2.0054) as a standard. Results were presented as the mean ±s.D. Student's t-test was used for statistical analysis and p
